Modulation of anxiety behavior by intranasally administered vaccinia virus complement control protein and curcumin in a mouse model of Alzheimer's disease.

Widespread neuroinflammation in the central nervous system (CNS) of Alzheimer's disease (AD) patients, involving pro-inflammatory mediators such as complement components, might be responsible for AD associated behavioral symptoms such as anxiety. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are the bioactive compounds of natural origin shown to inhibit the in-vitro complement activation. In order to develop complement regulatory compounds which could be delivered to the CNS by a non-invasive route, VCP, its truncated version (tVCP), and Cur were administered to Wistar rats intranasally. The distribution of these compounds in cerebrospinal fluid (CSF) was studied using an enzyme linked immunosorbent assay (ELISA), using VCP and tVCP as antigens and a modified fluorimetric method (Cur). VCP and tVCP were also detected in the olfactory lobes of the rat brain using immunohistochemical analysis. These compounds were then compared for their ability to attenuate the anxiety levels in APPswePS1δE9 mice using an elevated plus maze (EPM) apparatus. VCP treatment significantly improved the exploratory behavior and reduced the anxiety behavior in APPswePS1δE9 mice. tVCP however showed an opposite effect to VCP, whereas Cur showed no effect on the anxiety behavior of these mice. When these mice were subsequently tested for their cognitive performance in the Morris water maze (MWM), they showed tendencies to collide with the periphery of the walls of MWM. This unusual activity was termed "kissperi" behavior. This newly defined index of anxiety was comparable to the anxiety profile of the VCP and tVCP treated groups on EPM. VCP can thus be delivered to the CNS effectively via intranasal route of administration to attenuate anxiety associated with AD.

Vaccinia virus complement control protein is capable of protecting xenoendothelial cells from antibody binding and killing by human complement and cytotoxic cells.

BACKGROUND: Vaccinia virus complement control protein (VCP) was the first secretory microbial protein shown to have structural similarity to the family of complement control proteins. VCP can block both the classical and alternate complement pathways. Recently, VCP has been shown to bind to heparin, and this property contributes to separate functions, making the molecule a multifunctional protein. METHODS: VCP prepared from a natural infection of RK-13 cells with vaccinia virus was purified to homogeneity. Cultured pig aortic endothelial cells (PAECs) were mixed with human serum, anti-Gal alpha1,3 Gal antibody, neutrophils, or natural killer (NK) cells in the presence or absence of VCP and either direct binding of FITC-labeled antibody or killing by cytotoxic cells was estimated. RESULTS: Our cell culture studies demonstrate that VCP blocks complement-mediated killing of PAECs by human serum in a dose-dependent manner. We also demonstrate that VCP is capable of blocking Gal alpha1,3 Gal binding sites on PAECS. Surprisingly, VCP effectively blocked interactions between PAECs and cytotoxic cells such as human naive neutrophils and NK cells. CONCLUSION: VCP is a novel protein amongst the complement control protein family and can, not only block xenorejection by inhibiting complement but also by blocking killing by cytotoxic cells.

Approaches of the diagnosis of hepatitis viruses.

Hepatitis virus infection occurs in close to a billion people worldwide at some point in their lifetimes. Hepatitis B and C viruses together account for infections in half a billion people and are considered the most carcinogenic of any known biological agent. The diagnostic approaches to detect hepatitis viruses are discussed.

Examination of the interactions of the amyloid precursor protein carboxyl terminus to intracellular protein: possible role in apoptosis.

1. The carboxyl terminus of the amyloid precursor protein (APP) has several identified regions that may potentially contribute to the pathogenic effects of Alzheimer's disease (AD). To examine these effects, the authors iodinated a short synthetic peptide corresponding to amino acids 679-687 of APP695. They also produced a [35S]-Methionine labeled peptide corresponding to the entire carboxyl 100 amino acids of APP695 via a reticulocyte lysate coupled in vitro transcription/translation system. 2. Human neuroblastoma cells (SK-N-SH) and non-neuronal epithelial cells (RK-13) were cultured, harvested, and lysed. The S1 cell extract fractions were combined with either of the labeled peptides and incubated at different temperatures to allow for interaction and binding of cellular proteins with the peptides. These interactions were identified as gel mobility shift patterns on native PAGE gels. The presence of distinct bands on the gels indicate that the APP C-terminus interacts with several intracellular proteins, some of which may be detrimental to the cell. The authors have tested the possibility that the accumulation of C-terminal proteins may result in apoptosis. 3. Apoptosis in neural cells is one detrimental effect that has been attributed to APP. The authors examined hippocampal tissue sections from Alzheimer's disease (AD) and age-matched normal control patients for a difference in the number of apoptotic nuclei present using an in situ apoptosis detection kit that labels the numerous free DNA ends present in apoptotic nuclei. The number of apoptotic nuclei found in AD neuronal tissue was significantly higher than in normal tissue.

Dextran sulfate inhibits IFN-gamma-induced Jak-Stat pathway in human vascular endothelial cells.

Human vascular endothelial cells can be induced by IFN-gamma to express class II MHC proteins. Previously, dextran sulfate was shown to selectively inhibit expression of class II MHC by preventing transcription of the gene encoding CIITA, a transactivator protein required for IFN-gamma-inducible expression of class II genes. In this study we characterized the effects of dextran sulfate on the intracellular events occurring prior to CIITA activation. Immunoprecipitation and Western blot analyses indicated that IFN-gamma-induced phosphorylation of Stat1 and Jak2 was blocked by dextran sulfate. In addition, electron micrographs showing the large accumulation of dextran sulfate particles in the cytoplasms of endothelial cells demonstrated that Stat and Jak proteins may directly interact with dextran sulfate. Binding of radiolabeled IFN-gamma to cells indicated that dextran sulfate may also modulate IFN-gamma interactions with the cell surface. Thus, dextran sulfate is capable of interfering with the IFN-gamma-induced expression of class II MHC genes at multiple sites.

Detection of the membrane-retained carboxy-terminal tail containing polypeptides of the amyloid precursor protein in tissue from Alzheimer's disease brain.

A major hallmark of Alzheimer's disease (AD) is the presence of extracellular amyloid plaques consisting primarily of amyloid beta peptide (A beta) which is derived from a larger beta-amyloid precursor protein (APP). APP is processed via secretory and endosomal/lysosomal pathways by a group of proteases called secretases. During the processing of APP, the carboxy-terminal tail fragment has been suggested to remain within the cell. To investigate the fate of this fragment, we generated an antibody specific for a nine amino acid residue, the sequence of which was derived from the carboxy-terminal putative cytoplasmic tail of APP. Computer analysis of the entire APP gene, searching for regions of greatest antigenicity, surface probability, hydrophilicity, and presence of beta turns, indicated that the cytoplasmic tail region is an immunodominant region of APP. The peptide coupled to keyhole limpet hemocyanin protein, produced a very high titer antibody (1:1 x 10(6)). To evaluate the specificity of the antibody, immunoprecipitation of in vitro transcribed and translated DNA encoding the carboxy-terminal amino acids of APP in wheat germ extract was carried out. A single immunoprecipitated band of the correct size was seen by SDS-PAGE. The antibody was also able to specifically detect the accumulation of the stable C-terminal tail containing fragments of APP in neurites of the amygdala and hippocampus regions of the human brain tissue from AD subjects, but did not react with age-matched control normal brain tissue. The localization of the C-terminal tail of APP within the brain tissue of AD patients underscores the likely importance of the C-terminus in the pathogenesis of AD.

The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins.

Sequencing and computer analysis of the left (52,283 bp) and right (49,649 bp) variable DNA regions of the cowpox virus strain GRI-90 (CPV-GRI) has revealed 51 and 37 potential open reading frames (ORFs), respectively. Comparison of the structure-function organization of these DNA regions of CPV-GRI with those previously published for corresponding regions of genomes of vaccinia virus, strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR); and variola major virus, strains India-1967 (VAR-IND), Bangladesh-1975 (VAR-BSH); and alastrim variola minor virus, strain Garcia-1966 (VAR-GAR), was performed. Within the left terminal region under study, an extended DNA sequence (14,171 bp), unique to CPV, has been found. Within the right region of the CPV-GRI genome two segments, which are unique to CPV DNA (1579 and 3585 bp) have been found. Numerous differences have been revealed in the genetic structure of CPV-GRI DNA regions, homologous to fragments of the genomes of the above-mentioned orthopoxvirus strains. A cluster of ORFs with structural similarity ot immunomodulatory and host range function of other poxviruses have also been detected. A comparison of the sequences of ORF B, crmA, crmB, crmC, IMP, and CHO hr genes of CPV Brighton strain (CPV-BRI) with the corresponding genes in strain GRI-90 have revealed an identity at the amino acid level ranging from 82 to 96% between the two strains. The findings are significant in light of the recent demonstration of CPV as an important poxvirus model system to probe the precise in vivo role(s) of the unique virally encoded immunomodulatory proteins. Also, the presence of a complete and intact repertoire of immunomodulatory proteins, ring canal proteins family, and host range genes indicates that CPV may have been the most ancient of all studied orthopoxviruses.

Pro-inflammatory complement activation by the A beta peptide of Alzheimer's disease is biologically significant and can be blocked by vaccinia virus complement control protein.

The amyloid plaque is the hallmark of Alzheimer's disease (AD). The transmembrane domain and a portion of the C-terminus (A beta) of the amyloid precursor protein, are known to form the nucleus of the amyloid plaque. It has been demonstrated recently, using in vitro assays, that the A beta peptide can activate both the classical (antibody-independent) and alternate pathways of complement activation. The proposed complement activation is due to the binding of A beta to the complement components C1q and C3, respectively, which initiate formation of the proinflammatory C5a and C5b-9 membrane attack complex. In this report, we have investigated the in vitro findings for the likely complement-dependent proinflammatory properties of the Alzheimer's disease A beta peptide. We have performed experiments using congenic C5-deficient and C5-sufficient mice injected with synthetic A beta and recombinant polypeptide (C-100) containing A beta. Injection of C-100 into C5-sufficient mice induced a clear increase in the number of polymorphonuclear cells (neutrophils) at the site of injection due to complement activation and the subsequent release of proinflammatory chemtoactic factors. In sharp contrast, the C5-deficient mice did not show any increase in cellular influx. The vaccinia virus complement control protein, an inhibitor of both the classical and alternate pathway can down-regulate the biologically significant activation of complement by A beta, as demonstrated by an in vitro immunassay. The therapeutic down-regulation of A beta-caused complement activation could greatly alleviate the progression of some of the chronic neurodegeneration characteristic of Alzheimer's disease.

Microorganisms and their interaction with the immune system.

Microorganisms interact with the immune system in multiple ways. In an interaction between a microorganism and its host, the defense of the host does not go unchallenged. Microorganisms have for decades been suspected of possessing the capabilities of hiding from and escaping the consequences of immune surveillance. Escape mechanisms like antigenic variation, latency, and genomic integration can best be described as passive mechanisms for avoiding interaction with the host immune system, to differentiate them from the more engaging and host-directed active mechanisms of interaction. Studies of the mechanism of direct entry of viruses (HIV, measles, and enteroviruses), bacteria (streptococci and staphylococci), and parasites (Leishmania and plasmodium) into immune cells like CD4+ T cells or macrophages, as reported very recently, indicate an even more aggressive mode of interaction. This aggressive mechanism of interaction with the components of the host immune system allows the microbe not only to block the normal function of immune components on the surface of immune cells from functioning, but also to obliterate a vital immune function, cellular immunity, causing immunosuppression, e.g. the depletion of CD4+ T cells due to the entry and replication of HIV. Collectively, microorganisms have evolved various mechanisms by which they can actively block almost any cellular, humoral, or systemic immune response. One general feature of the proteins that assist microorganism to immunomodulate and actively evade host defense is their structural and therefore functional similarity to the host proteins, which they effectively mimic. Understanding the different mechanisms by which microorganisms interact with the immune system can impact the design of live vaccines as well as the development of novel therapeutic immunomodulators that can provide medicine with powerful new tools to manage immune disorders, allograft rejection, remote multiple organ failure resulting from trauma, autoimmune diseases, etc.

Severe and prolonged inflammatory response to localized cowpox virus infection in footpads of C5-deficient mice: investigation of the role of host complement in poxvirus pathogenesis.

Poxviruses are a large, complex group of highly successful pathogens that cause disease in humans and other animals. They encode several proteins postulated to be involved in the evasion of host immunity and therefore serve as excellent models for understanding virus-host interaction during the early stages of viral infection. Vaccinia virus, the best characterized member of the poxviridae family, encodes a 35-kDa major secretory polypeptide termed vaccinia virus complement control protein (VCP), which is structurally related to the family of human and mouse complement control proteins. Members of the family of complement control proteins have been shown to inhibit complement-mediated opsonization of bacteria and induction of inflammatory and phagocytic responses in vitro. Insertional inactivation of the VCP gene results in attenuation of viral virulence in vivo. The role of host complement in the inflammatory response to poxvirus infection has not been systematically investigated. Prior to determining the role of VCP on inflammatory responses in vivo, we decided to investigate the role of host complement in the progression of viral infection. We have compared the effects of injection of cowpox virus, primarily a rodent virus, into footpads of congenic mice strains B10.D2/nSnJ (C5-sufficient) and B10.D2/oSnJ (C5-deficient). The effects of the injection were monitored macroscopically by measuring the specific swelling response immediately following primary injection and subsequently after reinfection and by histological examination of the stained sections of the footpads. Our results indicate that there is a significant variation in the primary response in the two different mouse strains to cowpox virus infection. The specific swelling response observed in measurements from the footpads of the B10.D2/oSnJ mice was significantly greater, persisted for a longer duration, and was accompanied by severe ulceration, edema, induration, and hemorrhaging. Reinjection of the footpads after a 3-month period, during which time the swelling had subsided and the footpad had fully recovered to its original size and appearance, showed no significant differences between the two strains. This strongly suggests that the host complement plays a significant role during the initial response to poxvirus infection.

Routine laboratory diagnosis of hepatitis C virus infection.

Hepatitis C virus (HCV) is the major cause of parenterally transmitted non-A, non-B hepatitis. The analysis of the genomic sequence of HCV has facilitated the development of a number of diagnostic assays for testing circulating antibodies in serum from patients with HCV infection. Besides the first-generation ELISA and RIBA, which employed the C100-3 non-structural polypeptide, second-generation tests employing both structural and non-structural polypeptides are being rapidly introduced. Several coded panels were employed in a comparative study of HCV-SP ELISA (utilizing a new synthetic peptide whose sequence was derived from the structural region) along with first- and second-generation tests. On the basis of the results, evidently antigens corresponding to the structural components of the virus are more sensitive and specific for the early detection of HCV antibodies than tests using non-structural epitopes. Additionally epitopes of the structural region elicit a very strong antibody response in laboratory animals. An example of one such application is the detection of HCV specific antigens in semen from patients diagnosed with non-A, non-B (NANB) hepatitis. Semen samples from 9 patients clinically diagnosed as having NANB hepatitis were tested by an ELISA using antibodies against HCV-specific structural antigens. The semen from all 9 patients had HCV-specific structural antigens in comparison to semen from 5 healthy donors. Semen from 5 of the 9 patients had significant levels of the HCV-specific antigen. This approach to detecting HCV antigens could, if rigorously tested, evolve into promising new assays for detecting HCV.

Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope.

An enzyme-linked immunosorbent assay (ELISA) was developed by using a synthetic polypeptide (SP) whose sequence was derived from the structural region of hepatitis C virus (HCV). Results of several coded panels of sera obtained from volunteer blood donors and patients with apparent non-A, non-B hepatitis and/or hepatitis B virus used in this ELISA were compared with those of a commercially available first-generation C-100 ELISA (using nonstructural HCV antigens), an experimental second-generation C-200/C-22 ELISA (using both structural and nonstructural HCV antigens), and recombinant immunoblot assays RIBA-I and RIBA-II. In the majority of cases, the results obtained with the HCV-SP ELISA correlated well with those obtained by RIBA-II and C-200/C-22 ELISA. In contrast, many samples that were repeatedly reactive in the C-100 ELISA results were nonreactive with RIBA and HCV-SP ELISA. In addition, HCV-SP detected HCV-specific antibody that appeared within a month of infection and coincided with the earliest increase in alanine aminotransferase. In summary, we have developed an ELISA based on a structural HCV synthetic polypeptide, HCV-SP, that has high specificity and sensitivity and is capable of detecting specific antibodies in the acute phase of HCV infection.

Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses.

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.

Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein.

The objective of this study was to identify and characterize the gene encoding a protein of approximately 12 kDa that is secreted from cells infected with the vaccinia virus. The absence of this protein from the medium of cells infected with a spontaneous deletion mutant (6/2) suggested that the open reading frame (ORF) was located within a 12,800-base pair segment near the left end of the genome (G. Kotwal and B. Moss, Nature (London) 335, 176-178, 1988). Antibody to the 12-kDa protein immunoprecipitated an appropriate size in vitro translation product of mRNA that hybridized to a DNA segment containing an ORF (N1L) that could encode a 13.8-kDa polypeptide. The similarity in the sizes of the in vitro translation product and the secreted protein was consistent with the absence of processing. Transcriptional analysis revealed major and minor early RNA start sites preceding the N1L ORF as well as a late RNA start site with an atypical TAAAAT sequence. The N1L gene was interrupted by replacing a segment of the ORF with the Escherichia coli beta-galactosidase gene. When two-dimensional polyacrylamide gel electrophoretic patterns of [35S]methionine-labeled proteins secreted from cells infected with parental and recombinant viruses were compared, a spot missing from the latter corresponded in molecular weigh and isoelectric point with that predicted from the N1L ORF. The latter analysis revealed the presence of other secreted proteins of similar molecular weight but different isoelectric points that also appear to map within the left end of the vaccinia genome. The recombinant virus was attenuated as judged by the increased intracranial LD50 for mice but nevertheless induced antibody and cytotoxic responses after intradermal and intraperitoneal injections. Relative to the parental virus, the recombinant was also more attenuated for immunodeficient nude mice, based on their survival time after infection.

Vaccinia virus encodes two proteins that are structurally related to members of the plasma serine protease inhibitor superfamily.

Nucleotide sequencing adjacent to the right inverted terminal repetition of the vaccinia virus genome revealed two genes encoding polypeptides that are structurally related to members of the plasma serine protease inhibitor superfamily (SPI). Inclusion in the superfamily is based on extensive amino acid sequence similarities as well as a consensus sequence adjacent to the active-site region near the carboxyl ends of the proteins. The genes designated SPI-1 and SPI-2 are located 10,000 and 17,000 base pairs from the right end of the genome, respectively. The predicted SPI-1 polypeptide is 11 amino acids longer than that of SPI-2, and the deduced masses are 40,471 and 38,125 daltons, respectively. Similarities between SPI-1 and SPI-2 are indicated by the percentage of identical amino acids (44%) and corresponding hydrophobicity plots. The maximum amino acid sequence diversity occurs precisely in the putative active-site region, suggesting that SPI-1 and SPI-2 may inhibit different proteases. SPI-2 is homologous to a previously described cowpox virus gene (D. J. Pickup, B. S. Ink, W. Hu, C. A. Ray, and W. K. Joklik, Proc. Natl. Acad. Sci. USA 83:7698-7702, 1986). Evidence for a cowpox virus homolog of SPI-1 was obtained by DNA hybridization. Thus, the presence of two genes that belong to the plasma serine protease inhibitor superfamily may be characteristic of orthopoxviruses.

Vaccinia virus encodes a secretory polypeptide structurally related to complement control proteins.

Several polypeptides are secreted into the medium of cells infected with vaccinia virus, a cytoplasmic DNA virus belonging to the poxvirus family. One of these, a polypeptide of relative molecular mass 19,000 is structurally related to epidermal growth factor and binds to epidermal growth factor receptor stimulating proliferation of uninfected cells in vitro and in vivo. Here, we show that a second, and much more abundant secretory polypeptide, is also encoded by vaccinia virus and is structurally related to the superfamily of complement control proteins. Members of this family can block complement-mediated induction of the inflammatory response, and engulfment, killing and lysis of bacteria and viruses.

Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein.

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.

Role of fatty acid acylation of membrane glycoproteins. Absence of palmitic acid in glycoproteins of two serotypes of vesicular stomatitis virus.

The presence of fatty acids bound to the glycoprotein in a number of serotypes of vesicular stomatitis virus was investigated by growing the virus in the presence of [3H]palmitic acid. [3H]Palmitate was efficiently incorporated into G proteins of the serotypes Indiana, Piry, and Chandipura but was not detected in G proteins from Cocal and three different strains of New Jersey serotypes. Cerulenin, an inhibitor of fatty acid acylation, also did not inhibit the maturation of Cocal serotype. These results suggest that fatty acid acylation of viral membrane glycoproteins may not be a general requirement for maturation and budding of enveloped viruses.

Viral membrane glycoproteins: comparison of the amino terminal amino acid sequences of the precursor and mature glycoproteins of three serotypes of vesicular stomatitis virus.

The NH2-terminal amino acid sequences of the envelope glycoproteins and the in vitro synthesized, nonglycosylated precursors of the glycoproteins of three serotypes, namely Indiana (Toronto), Cocal, and New Jersey (Concan) of vesicular stomatitis virus were determined. A comparison of the sequences showed little homology in the signal peptides present in the nonglycosylated precursors except for their high hydrophobic amino acid content. In contrast, the NH2-terminal amino acid sequences of the mature envelope glycoproteins revealed extensive homology suggesting that this region is conserved and may be involved in essential biological function(s) of the rhabdovirus.

Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography.

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.